Physiologically active polypeptides are useful as therapeutic agents for specific diseases but when they are administered into the blood, they are unstable and thus a sufficient pharmacological effect cannot be expected in many cases. For example, when a polypeptide having a molecular weight of about 60,000 or less is administered into the blood, it is filtered through glomerulus of the kidney and most of them is secreted into the urine, so that a remarkable therapeutic effect cannot be obtained even if it is used as a therapeutic agent. Thus, a repeated administration is frequently required. Moreover, other polypeptides may be decomposed by a hydrolase or the like existing in the blood to lose the physiological activity. Furthermore, even in the exogenous physiologically active polypeptides, the physiological activity may sometimes be effective for treating diseases. However, since the exogenous physiologically active polypeptides, polypeptides produced by genetic recombination and the like have a structure different from that of an endogenous polypeptide, it is known that the exogenous polypeptides may induce immunoreactions to cause serious side effects such as anaphylactic shock and the like. In addition, some physiologically active polypeptides may frequently be accompanied by the problem of physical properties such as poor solubility and the like when they are used as therapeutic agents.
As one method for solving the problems when physiologically active polypeptides are used as therapeutic agents, a method is known wherein at least one molecule of an inactive polymer chain is chemically linked to the polypeptides. In most cases, a desired property is imparted to the polypeptides or proteins by chemically linking a polyalkylene glycol such as polyethylene glycol or the like to the polypeptides. For example, in superoxide dismutase (SOD) modified with polyethylene glycol, the half-life in the blood is markedly prolonged and thus duration of the activity is found [Pharm. Research Commun., 19: 287 (1987)]. Moreover, the modification of granulocyte colony-stimulating factor (G-CSF) with polyethylene glycol is also known [J. Biochem., 115: 814 (1994)]. Furthermore, examples of polyethylene glycol-modified polypeptides such as asparaginase, glutaminase, adenosinedeaminase, uricase and the like are summarized by Gillian E. Francis et al. [Pharmaceutical Biotechnology, vol. 3, Stability of Protein Pharmaceuticals, Part B, p. 235 (1992), Plenum Press, New York]. Additionally, as the effect obtained by modifying physiologically active polypeptides with a polyalkylene glycol, increase of thermal stability [Biophysics (Seibutsubutsuri), 38: 208 (1998)], solubilization in an organic solvent [Biochem. Biophys. Res. Commun. (BBRC), 122: 845 (1984)] and the like are known.
On the other hand, examples of the method of linking a peptide or protein to a polyalkylene glycol include a method wherein an active ester of a carboxylic acid, a maleimido group, carbonate, cyanuric chloride, a formyl group, an oxiranyl group or the like is introduced into the terminal end of the polyalkylene glycol and then the product is linked to an amino group or a thiol group of a polypeptide [Bioconjugate Chem., 6: 150 (1995)]. These techniques include an example wherein the stability in the blood is enhanced by linking polyethylene glycol specifically to a particular amino acid residue in a polypeptide without decreasing the physiological activity of the peptide or protein. As modification with polyethylene glycol which is specific to a particular amino acid residue in a polypeptide, there are an example wherein polyethylene glycol is linked to the carboxy-terminal of a growth hormone-releasing factor through a spacer of norleucine [J. Peptide Res., 49: 527 (1997)], an example wherein cysteine is introduced into the 3-position of interleukin-2 by genetic recombinantion and then polyethylene glycol is specifically linked to the position [BIO/TECHNOLOGY, 8: 343 (1990)] and the like.
Most of the above polyalkylene glycol-modified polypeptides are obtained by the method of linking a linear polyalkylene glycol, but it has been found that a method of linking a branched polyalkylene glycol is excellent as a method for obtaining chemically modified polypeptides having a potent activity. It is known that a larger molecular weight of a polyalkylene glycol or a higher modification degree generally results in that duration in the blood is prolonged [The Journal of Biological Chemistry, 263: 15064 (1988)], but a high modification degree sometimes causes decrease of the physiological activity of the polypeptide. One reason of the decrease is that a particular amino group, a thiol group or the like necessary for the physiological activity in the polypeptide is modified with a chemically modifying agent. Interleukin-15 is known as an example wherein the physiological activity decreases depending on the modification degree [J. Biol. Chem., 272: 2312 (1997)]. On the other hand, with regard to polyalkylene glycols having a large molecular weight, it is difficult to synthesize those having a homogeneous molecular weight distribution and high purity. For example, in monomethoxy polyethylene glycol, contamination of a diol component as an impurity is known. Thus, it has been attempted to produce a modifying agent having a large molecular weight by branching highly pure polyalkylene glycols having a narrow molecular weight distribution available at present through a spacer. Thus, a chemically modified polypeptide having a potent physiological activity can be obtained with retaining durability even when the modification degree is reduced. Also, it is considered that the surface of a physiologically active polypeptide can be covered more efficiently by branching a polyalkylene glycol. For example, a double-chain polyethylene glycol derivative is known wherein cyanuric chloride is used as a group having a branched structure (Japanese Published Unexamined Patent Application Nos. 72469/91 and 95200/91). In this case, methoxy polyethylene glycol having an average molecular weight of 5,000 is used but there is fear of toxicity derived from a triazine ring in the compound. Moreover, Japanese Published Unexamined Patent Application No. 153088/89 discloses that a chemically modified polypeptide having a more potent activity can be obtained with a lower modification degree by using a comb-shaped polyethylene glycol which is a copolymer of polyethylene glycol and maleic anhydride as compared to a linear polyethylene glycol. However, a number of reaction sites with a polypeptide are present and the molecular weight distribution is not uniform in the compound. In addition, an example wherein polyethylene glycols are branched through a benzene ring using cinnamic acid as a starting material (Japanese Published Unexamined Patent Application No. 88822/91) is also known. In the above branched polyalkylene glycols through a triazine ring or a benzene ring, the structure at the branching point is plane, so that spatial movement of the polyalkylene glycol chain is restricted and thus they are considered to be disadvantageous for the effect of increasing the molecular size. As another example, a compound wherein two polyethylene glycol are branched using lysine as a branching point (WO 96/21469, U.S. Pat. No. 5,643,575) and the like are known, but the compound having branches at a cyclic structure of the present invention is unknown. Furthermore, the above conventional branched polyalkylene glycols achieve the increase of the molecular weight by linking at least two molecules of a polyalkylene glycol, but it is not known that the molecular size can be increased more effectively when physiologically active polypeptides are modified with a branched polyalkylene glycol than the case when they are modified with a linear polyalkylene glycol having the same molecular weight.
The branched polyalkylene glycols as a modifying agent which overcome the problems of the above conventional polyalkylene glycols, have a low toxicity and an improved stability and are excellent in the effect of increasing the molecular size have been desired.
Recently, an interferon-β preparation has been paid attention to as a therapeutic agent for multiple scleroses [The Lancet, 352(7): 1491 (1998) and the like]. Multiple scleroses are demyelinating autoimmune diseases of unknown etiology and are characterized in the infiltration of perivascular cell of central nerve white matter and successive destruction of myelin sheath. The mechanism of the outbreak is unknown but it is considered that a nerve conduction disorder occurs as a result of demyelination spots which are caused through the destruction of myelin sheath covering the axon of a central nerve by immunocyte and thereby various disorders of neural functions may be exhibited. In Western countries, interferon-β (IFN-β) becomes a mainstream therapeutic agent for multiple scleroses but the IFN-β1b preparation clinically used at present has problems that subcutaneous injection on alternate days is required and the like, so that a therapeutic agent which has an increased activity and is effective even at less number of dose frequency has been desired. Moreover, interferons are effective for viral hepatitis such as hepatitis C, hepatitis B and the like, and treatment of various cancers such as leukemia, lymphoma, myeloma, osteosarcoma, breast cancer, kidney cancer, brain tumor and the like, viral or inflammatory skin diseases, and eye diseases in addition to multiple scleroses [Pharmacy (Yakkyoku), 41(6): 769 (1990)], and the possibility as a therapeutic agent for diseases relating to vascularization is suggested [Tissue Culture (Soshikibaiyo), 22(7): 278 (1996)]. For these diseases, an effective therapeutic agent having a high duration at a low dose and a low dose frequency has been desired as well.